Specialized metabolic sensing neurons in the arcuate (ARC) and ventromedial hypothalamic nucleus (VMN) utilize both glucose and long chain fatty acids (LCFA) as signaling molecules to alter their activity. These neurons are known to be involved in the regulation of the counterregulatory responses (CRR) to insulin- induced hypoglycemia (IIH), energy and glucose homeostasis in the body. We have identified glucokinase (GK) as a neuron-specific regulator of neuronal glucosensing and the FA receptor/transporter CD36 as a regulator of neuronal FA sensing. Manipulating GK activity in the ventromedial hypothalamus ((VMH: ARC + VMN) markedly alters the CRR to IIH and glucoprivic feeding but lowering it had no effect on energy homeostasis or glucose tolerance in rats fed low, low sucrose diets. However, rats selectively bred to develop diet-induced obesity (DIO) on a 31% fat, high sucrose, high energy (HE) diet have an inborn elevation of ARC GK in association with altered numbers of glucose and FA sensing VMN neurons, hyperphagia when exposed to HE diet and a blunted CRR to IIH as compared to selectively bred diet-resistant (DR) rats. Using DIO, DR and outbred rats and mice with fluorescently tagged neuropeptide Y (NPY) and proopiomelanocortin POMC) neurons or CD36 deletion, we will utilize calcium imaging, quantitative PCR, in vivo assessment of CRR, energy and glucose homeostasis to pursue the Overall Hypothesis that GK and CD36 are critical regulators of metabolic sensing in ARC and VMN neurons and that sensing of these substrates is critical for the modulation and blunting of the CRR to IIH, the development of hyperphagia and obesity on HE diet and glucose production and disposal. Aim 1 will assess the role that GK plays in regulating glucosensing in identified ARC NPY and POMC neurons in mice and DIO and DR rats and assess the effect of lowering VMH GK activity to prevent the blunted CRR following repeated bouts of IIH. Also, since selective serotonin reuptake inhibitors increase the CRR and counteract its blunting, we will investigate the role of VMH serotonin in regulating the CRR and the expression of GK. Aim 2 will assess the role of VMH GK in the regulation of sympathetic activity, hepatic glucose production, energy homeostasis and development of obesity in DIO vs. DR rats by altering its expression with adenoviral vectors. Aim 3 will investigate the mechanisms by which ARC neurons sense various LCFA and use adeno-associated viral vectors to reduce the VMH expression of CD36, a LCFA receptor-transporter that we have shown regulates 50% of neuronal FA sensing, to assess the effects on both the CRR to IIH and the development of obesity in DIO vs. DR rats on high fat, high sucrose diets. The overall goal is to identify molecular targets involved in the regulation of glucose and FA sensing, that also affect energy and glucose homeostasis, as potential therapeutic targets to prevent blunting of the CRR in diabetics and the development of obesity and insulin resistance in humans on high fat, high sucrose diets.